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The results show that enzyme I of gramicidin S synthetase activates D-kUCine in a manner analogous to that of L-leucine (ATP + D-leucine + D-leucyl-adenylate + PPi) and binds two moles of D-leucine in thioester linkage. It has therefore two thiol sites for D-leucine, compared to one for the L-isomer. One of the sites seems to be the thiol site for L-leucine which is 1 herefore stereo-unspecific. The results also indicate that the site for the formation of the aminoac) 1 adenylates is the same for the two isomers. I n contrast to thioester-bound L-leucine, enzyme-bound D-leucine does not catalyze the ATP-[14C]AMP exchange reaction.At a molar ratio of L-leucine : D-leucine equal to 4, gramicidin S synthesis is almost completely inhibited and the growth of the peptide chain stops a t the tetrapeptide stage (D-Phe-L-Pro-I,-Val-L-Om). Enzyme-bound D-leucine is thus not able to replace L-leucine in the formation of the pentapeptide.The biosynthesis of the cyclic decapeptide gramicidin S which has the following amino acid se-has been the subject of investigation in several laboratories and a fair amounts is now known about this non-ribosomal peptide synthesis [i].At an early stage in our study with a crude gramicidin S synthetase preparation, it was found that the addition of D-leucine to the enzyme reduced the incorporation of ~-[l~C]leucine into gramicidin. The results could be explained by the presence of a leucine racemase or by an inhibition of gramicidin S synthesis. This finding has now been investigated in the hope of elucidating further details about the biosynthesis of gramicidin S .The present work shows that D-leucine is an inhibitor of gramicidin S synthesis, and that synthesis stops a t the tetrapeptide stage. D-hUCine like L-leucine is activated by the formation of the aminoacyl adenylate and then bound to the enzyme in a thioester linkage. The activation site seems to be the same for the two isomers since they show competitive kinetics in the ATP-[32P]PP~ exchange reaction. The results further suggest that the enzyme binds two moles of D-leucine whereas it only binds one mole of L-leucine. One of the thiol sites involved in the binding of D-leucine is probably the site which binds L-leucine. It is suggested that the other thiol site for D-leucine could be the postulated "waiting site" for the pentapeptide [3].A brief report of these results has been published elsewhere 141. METHODS AND MATERIALS Labelled Substances Gramicidin-X Xynthetasta Assay of Gramicidin-X SynthesisThe incubation mixture and the procedure for isolating gramicidin S were as described previously

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